Glycoproteins are integral components of the plasma membranes of animal cells. While the addition of the terminal glycose moieties to these proteins has been shown to occur by stepwise transfer from a sugar-nucleotide donor to the appropriate acceptor, neither the discrete steps involved in the biosynthesis of the core structure nor the transfer of this core to the protein are yet understood. Lipid-linked saccharides have recently been implicated as intermediates in the biosynthesis of the glycose core region. I propose to isolate and characterize mutants of CHO cells defective in the synthesis and transfer of this glycose core. These mutants will be obtained by the following three procedures. First, since the lectin concanavalin A binds to alpha-mannosyl residues, which are components of the core of membrane glycoproteins, CHO cell lines resisting to this lectin will be examined in vitro for defects in glycosylation. I will collaborate with Dr. R.M. Baker and Dr. Maria Cifone in these studies. Second, mutagenized clones will be screened for those which do not produce infectious Sindbis and Vesicular stomatitis virus (VSV). The envelopes of both these lytic viruses contain glycoproteins synthesized by cellular transferases; these glycoproteins have been implicated in viral infectivity. Third, cell lines resistant to infection by VSV will be examined; glycose-containing macromolecules have been shown to bind enveloped viruses in vitro. These studies are planned in collaboration with Dr. Bob Tabor. The mutants isolated by these last two methods will be subjected to a rapid biochemical screening procedure to identify those defective in incorporation of a core sugar into glycoproteins. All such glycosylation mutants will be examined biochemically to determine the specific lesion in the biosynthesis of their membrane glycoproteins.